JP2009530365A - Magnolia champaca oil, process for its preparation and composition containing it - Google Patents

Abstract

  The present invention relates to an oil extracted from at least one magnolia champaca flower, a method for its preparation, a cosmetic, pharmaceutical or dermatological composition comprising it and the prevention and / or treatment of harmful changes in the skin. Of its use as a multifunctional active ingredient.

Description

  The present invention relates to an oil extracted from at least one magnolia champaca flower, a process for its preparation, a cosmetic, pharmaceutical or dermatological composition comprising it, as well as physiological mechanisms or mechanisms relating to aging or aging in particular. It relates to its use as a multifunctional active ingredient in pharmaceutical, cosmetic or dermatological compositions for preventing and / or treating harmful changes in the skin due to diseases related to.

  The skin is mainly composed of three layers: the epidermis, the dermis and the subcutaneous tissue, starting from the outermost part.

  The epidermis, which is the outer layer of the skin, is layered and greatly contributes to providing protection against attacks from the outside world. The dermis is a connective tissue that provides both connective and skin nourishing functions.

  Skin aging occurs as a result of two separate and independent processes involving intrinsic or extrinsic factors.

  Endogenous aging or temporal biological aging corresponds to age-related “normal” or physiological aging.

  Exogenous aging generally corresponds to aging brought about by the environment, more particularly photoaging by exposure to the sun.

  The present invention relates to endogenous or physiological skin aging and exogenous skin aging.

  Skin aging results from alteration of connective tissue and a decrease in cell regeneration ability. This effect becomes apparent with the appearance of fine lines and spots over time. Microcirculation is reduced in the surface layer of the dermis. Macromolecules such as collagen, elastin and glycosaminoglycan, which are composed of hyaluronic acid as a constituent component, are chemically modified. The actual thickness of the dermis is reduced, the fibers are degraded, and the skin loses its biomechanical properties and elasticity. The phenomenon of chemical and enzymatic oxidation becomes more pronounced with aging and leads to an increase in cross-linking reactions between fibers such as collagen fibers.

Detrimental changes associated with aging can appear in a variety of ways, including the following:
-Unorganization of elastin fibers resulting in loss of stiffness, flexibility and elasticity or onset of telangiectasia;
-Loss of brightness due to reduced microcirculation and slowing of cell turnover in the epidermis and the appearance of fine lines or fine lines;
-Yellowing of the skin expressing a parchment-like appearance with the appearance of pigmented stains associated with dysfunction in melanin synthesis (ie melanogenesis);
-Skin dryness due to reduced barrier function of the stratum corneum and slowing of epidermal alternation.

  For these reasons, multifunctional activities that can have an effect on the combination of causes that cause deleterious changes to the skin by aging and / or by changes in physiological mechanisms associated with aging or the like There is a need to provide ingredients.

  Magnolia champaca, also known in the literature as Michelia champaca, is a shrub belonging to the family Magnoliaceae, native to the humid forest regions of China. The flower essential oil is commonly used in perfumes.

  Patent application JP 07/061918 discloses a cosmetic composition comprising a Michelia champaca extract obtained by extraction with a polar solvent and its use as an antioxidant.

  Michelia champaca L. has whitening effect on skin, scavenging active oxygen or antibacterial activity. A composition comprising the extract of is disclosed in patent application JP-A-2000 / 095663.

  Patent application JP-A-2005 / 068075 relates to a UV-preventing composition that absorbs UVA and UVB light and comprises an aqueous extract of Michelia champaca plants.

  Michelia Champaca L. The anti-inflammatory and antipyretic activity of the methanol extract of (White Variety) Vimala et al., Indian J. et al. Exp. Biol. 1997, 35, 1310-1314.

  The authors of the present invention are now quite surprising that oil extracted from at least one magnolia champaca flower is associated with aging or aging in the epidermis and / or dermis via induction or inhibition of physiological mechanisms. Have demonstrated an activity that can have an effect on symptoms due to physiological mechanisms or diseases associated with these mechanisms.

  This observation has an effect on symptoms due to new cosmetic, pharmaceutical or dermatological compositions, more particularly physiological mechanisms associated with aging or aging in the epidermis and / or dermis or diseases associated with these mechanisms Has been developed for use in all desired applications.

  The term “oil” is understood to mean a substance obtained by extraction with a non-polar solvent and very rich in water-insoluble compounds.

Therefore, the present invention, according to the first aspect,
20 to 70% benzoic acid esters,
15 to 35% saturated and / or unsaturated fatty acids (the ratio is expressed as a relative percentage of the sum of the components separated by gas chromatography)
It is related with the oil extracted from the flower of at least 1 type of magnolia champaca characterized by including these.

  Preferably, the oil of the present invention is derived from magnolia champaca, also known as “gold flower”, variety gold.

  The term “content expressed as a relative percentage of the sum of the components separated by gas chromatography” means the content of the individual components determined relative to the sum of the components separated by the gas chromatography system. Understood. There are only compounds that are extracted by the solvent during sample preparation and can be evaporated in the injector.

  Samples are from French Standard NF T 60-233, May 1977, “Preparation des esters methyles d'acides glass” (§ 5.2-Method applicable aux corps acids acid methyl esters acid fatty acids). Preparation "(§5.2-Methods applied to acid fatty substances and fatty acids)]. Chromatographic conditions are described in French standard NF EN ISO 5508, June 1995.

  Briefly, the method consists in esterifying the fatty acid in an acidic methanolic medium, then extracting with heptane, and then injecting the heptane solution into gas chromatography.

Preferably, the oil of the present invention is:
-10 to 40% cinnamyl benzoate,
-5-30% phenylethyl benzoate,
5 to 30% benzyl benzoate,
-5 to 30% linoleic acid,
-2 to 20% linolenic acid (the ratio is expressed as a relative percentage of the sum of the components separated by gas chromatography)
including.

  The invention also relates, according to a subsequent aspect, to a process for preparing magnolia champaca oil as defined above comprising at least one molecular distillation step.

  The starting material used consists, for example, of freshly picked flowers (fresh flowers) of at least one magnolia champaca, preferably magnolia champaca variety gold. The flowers are usually crushed or chopped.

Flowers are subjected to extraction with at least one non-polar solvent such as hexane, cyclohexane, heptane, isooctane, supercritical CO ₂ or dichloromethane.

  Extraction is generally performed by immersing or gently stirring the flowers in one or more of the above solvents, for example, in the temperature range from ambient to 100 ° C. for about 30 minutes to 12 hours.

  After filtration, the extraction may be repeated several times. The solution is filtered together to remove insoluble parts of the flower. Where appropriate, if the solvent is a volatile solvent such as hexane or heptane or cyclohexane, the solvent is also removed.

  This extraction step is conventional in the field of plant extracts and the person skilled in the art is in a position to adjust its reaction parameters based on his general knowledge.

  According to an advantageous embodiment of the invention, the fractionation step may be carried out to purify the product obtained as a result of the extraction.

C ₁ -C ₄ alcohols, such as methanol, ethanol, such as isopropanol and the like, polyols such as propylene glycol, dipropylene glycol, or ethyl acetate, hexane, an organic solvent such as cyclohexane, or common in the art It may be preferred to use a solvent or mixture of solvents selected from any other solvent.

Supercritical fluid, preferably by fractionation also be used with supercritical CO _2.

  As noted above, if one or more volatile solvents are used, there will be a basis for removing the volatile solvent before proceeding to the next stage.

  Magnolia champaca coagulum is obtained as a result of the extraction and optionally the fractionation steps described above. This type of extract is usually used as an intermediate in the manufacture of absolutes intended for the perfume industry.

  Advantageously, the coagulum is subjected to a molecular distillation step performed in a wiped film and a short process distillation apparatus.

According to a preferred embodiment, the present invention thus provides:
-Extraction from at least one magnolia champaca flower (preferably fresh flowers) with at least one non-polar solvent;
-Relates to a process for the preparation of magnolia champaca oil as defined above, comprising: subjecting said extract to at least one molecular distillation stage; and-recovering the distillate.

  It is preferred to use a wiped film and a short process molecular still in the performance of the molecular distillation stage. These devices include a distillation chamber that provides a rotating wiper device that allows the material to be distilled to be continuously spread over the distillation surface (hot surface). The substance vapor is concentrated using a cooling finger located in the center of the distillation chamber. Residues and distillates are collected by gravity flow. The purpose of this approach is to separate the components of a complex mixture by taking advantage of their boiling point differences. Wiped film and short-step molecular distillation have the advantage of lowering the distillation temperature by the distillation being carried out under high vacuum and also reducing the residence time of the mixture separated in the still. The degradation rate of the substance increases dramatically as a function of temperature and exposure time, and in an “alembic” type still, for example, the mixture may be held at elevated temperatures for hours, which causes denaturation .

  To date, in the case of vegetable oils, such methods have only been used for the separation of unsaponifiable fractions or for purification of the vegetable oils by removing the unsaponifiable fractions, or also for water-soluble or fat-soluble fractions. Has been used only for the decolorization or deodorization of the extract.

  In fact, it has now surprisingly been found that the use of a molecular distillation step now makes it possible to separate an oily fraction from a fat-soluble extract in which a small amount of the oily fraction is present.

  Preferred conditions for carrying out this method are shown below.

  Advantageously, prior to distillation, a heavy solvent selected from mineral or vegetable oils and polyols such as polyethylene glycol, for example, after extraction and optionally fractionation, to facilitate distillation or flow along the column. Add to the resulting magnolia champaca coagulum.

  The mixture of coagulum and heavy solvent obtained after extraction and optionally fractionation is heated at a constant flow rate at a temperature between 20 ° C. and 120 ° C., preferably between 50 ° C. and 100 ° C. Installed in the hot wall of the cylinder type evaporator.

  By spreading with a rotating annular wiper, the mixture is covered so as to cover the entire surface of the hot wall maintained at a temperature between about 100 ° C. and 200 ° C., preferably between 110 ° C. and 170 ° C. Spread it into a thin film.

  The volatile material is then contacted with the hot wall and while the less volatile material flows along the wall at a very low pressure of about 10 mbar to 0.001 mbar throughout the evaporator. Evaporates partially and gradually.

  The generated steam is preferably between 40 ° C. and 120 ° C., in particular between 60 ° C. and 100 ° C., on a cold wall that is concentric with the hot wall and located very close to the hot wall. It is concentrated at the temperature of

  The material separated during the operation of the device flows along the hot and cold walls by gravity.

  The distillate is collected and, if appropriate, subjected to additional separation operations (eg filtration or centrifugation).

  In this way, the magnolia champaca oil of the present invention is obtained.

  Advantageously, the oil is light colored. Furthermore, the oil does not contain solvents or any other chemical reactants that are involved during this extraction.

  The oils can also be obtained by standard methods without causing formulation problems related to the concentrations normally required to obtain activity in cosmetic, pharmaceutical or dermatological compositions in emulsion form. In contrast to when the resulting plant extract is in a concentrated form, it is provided in a fully concentrated form that can be used without showing a dark color.

  For this reason, the oils of the invention can be used directly in the preparation of cosmetic, pharmaceutical or dermatological compositions.

  The present invention also relates to magnolia champaca oil obtainable by the method described above.

  According to a subsequent aspect, the present invention is a multifunctional active ingredient for preventing and / or treating harmful changes in the skin due to aging or physiological mechanisms associated with aging or diseases associated with these mechanisms. Of magnolia champaca oil as described above in cosmetic, pharmaceutical or dermatological compositions.

  In particular, the invention relates to agents that improve healing or cell repair and / or agents that activate epidermal cell metabolism and / or agents that trap free radicals and / or agents that detoxify the epidermis. The use of the above-described magnolia champaca oil in a cosmetic composition.

  The invention also provides agents that improve healing or cell repair and / or agents that activate epidermal cell metabolism and / or agents that trap free radicals and / or agents that protect nuclear DNA and Also relates to the use of magnolia champaca oil as described above for the preparation of a pharmaceutical or dermatological composition as an agent for protecting mitochondrial DNA and / or as an agent for detoxifying the epidermis. .

  This is because the magnolia champaca extract of the present invention has been found to have some beneficial activity with respect to the prevention or repair of physiological mechanisms especially related to harmful skin changes due to aging. It is.

  Accordingly, the present invention more particularly relates to the use of magnolia champaca oil as described above in cosmetic, pharmaceutical or dermatological compositions as an agent inhibiting melanin synthesis, in particular as an agent inhibiting endothelin synthesis. Regarding use. Endothelin is a messenger protein that promotes melanocyte proliferation and increased melanogenesis, which causes skin pigmentation associated with aging (“liver spots”).

  The present invention particularly relates to the above-mentioned Magnolia champaca oil in cosmetic or dermatological compositions as an agent that inhibits the transcription factor MITF (microphthalmia-related transcription factor), a factor that regulates and differentiates melanocytes. Regarding use.

  Advantageously, it has also been found that the magnolia champaca oil of the present invention stimulates the synthesis of several families of growth factors by keratinocytes.

In particular, this activity is exerted on the following growth factors:
-Exerts mitogenic activity especially on the majority of cells derived from mesenchyme (Lepisto J. et al., 1995, Biochem. Biophys. Res. Commun., 209 (2), 393-9), and Stimulates the synthesis of collagen and collagenase by these cells, thereby playing a role in physiological methods such as healing and tissue repair (Tan EM et al., Biochem. J., 1995, 310 (Pt 2) , Pp. 585-8) PDGF, a platelet-derived growth factor; the level of PDGF induction and the ability of cells to replicate has also been shown to be age related: senescent fibroblasts have reduced levels of PDGF (Karlsson C. et al., J. Cell Physiol., 1994, 158 (2), 256. 62 pages). It has also been shown that the amount and type of various growth factors was able to explain the age-related differences in the ability of tissues to repair themselves (Ashcroft GS et al., J. Anat., 190, 1997 (Pt3), 351-65).
-In the skin, VEGF corresponding to the main skin angiogenic factor, namely vascular endothelial growth factor. The epidermis is an important source of VEGF that is secreted in large quantities by the growth of keratinocytes. VEGF mRNA is expressed by normal keratinocytes both in tissue and in cell culture. VEGF has been shown to maintain the ability to respond to stimulation of endothelial cell homeostasis and endothelial cell vasculature, even in elderly subjects (Watabebe Y. et al., 1997, Oncogene, 14 , 2025-2032).

Furthermore, a decrease in VEGF has been observed following exposure to UV radiation (Mildner M. et al., Photochem. Photobiol., 1999, 70 (4), 674-9).
-Plays an important role in the regulation and differentiation of keratinocytes (Iwamoto et al., Cytokine and Growth Factors Reviews, 2000, 11, 335-344) and its growth is dependent on this factor in aging of young cells (JID Suppl., 24, S46-S50; Kanzaki Y. et al., Exp. Cell. Res., 2002, 279 (2), 321-329), HB-EGF, a heparin-binding epidermal growth factor.

  The term “activator of the synthesis of several families of growth factors”, according to the invention, refers to the activity of at least one of these growth factors expressed and / or synthesized by and in keratinocytes and / or It is understood to mean any molecule that can stimulate expression and / or release.

  The present invention therefore provides cosmetic, as an agent that activates the synthesis of at least one growth factor by keratinocytes, in particular as an agent that activates at least one growth factor selected from PDGF, VEGF and HB-EGF. The use of magnolia champaca oil as described above in a pharmaceutical or dermatological composition.

  The present invention also provides the above as an agent that inhibits the activity of enzymes associated with matrix degradation, in particular matrix metalloproteinase (MMP), very particularly MMP3 (ie stromelysin 1) and MMP9 (ie gelatinase B). It also relates to the use of the described magnolia champaca oil.

  Matrix metalloproteinases are enzymes (endoproteases) that degrade the extracellular matrix in the context of physiological remodeling of the skin. Aging and exposure to A and / or B type UV radiation increases the activity of these MMPs, especially MMP3 (ie stromelysin 1) and MMP9 (ie gelatinase B), which increases cell proliferation. Deceleration results in aging due to aging of the skin (WO 98/36742). For this reason, the extracellular matrix undergoes even more significant degradation, resulting in the formation of skin tissue sagging and wrinkles (Rittie L. et al., Ageing Res. Rev., 2002, 1 (4 ), 705-20; Chung JH et al., J. Invest. Dermatol., 2001, 117 (5), 1218-24).

  The term “metalloproteinase inhibitor” according to the invention is any molecule capable of inhibiting the activity and / or expression and / or release of at least one metalloproteinase expressed and / or synthesized by and in the skin. Is understood to mean.

  The invention also relates, according to a preferred embodiment, to the use of magnolia champaca oil as described above, as an antioxidant, in a cosmetic composition or for the preparation of a pharmaceutical or dermatological composition. .

  The present invention also, according to a preferred embodiment, of the magnolia champaca oil as described above, as an agent that stimulates cellular metabolism, in cosmetic compositions or for the preparation of pharmaceutical or dermatological compositions. Also related to use.

  The present invention also, according to a preferred embodiment, by stimulating the synthesis of an agent for detoxifying the epidermis, in particular DT diaphorase (NQO1), which catalyzes the detoxification of quinones, and chemical metabolism such as lipid peroxidation products. In the preparation of a pharmaceutical or dermatological composition as a drug for the detoxification of epidermis subjected to oxidative stress, by stimulating the synthesis of glutathione S-transferase that catalyzes the detoxification of food For the use of the above-mentioned magnolia champaca oil.

  The present invention also, according to another preferred embodiment, as an agent that stimulates cellular energy, in particular cytochrome C and AMP kinase, in cosmetic compositions or for the preparation of pharmaceutical or dermatological compositions Also, the use of the magnolia champaca oil described above.

  Cytochrome C oxidase is the final step in the electron transport chain, is critical for cellular respiration, and also contributes to the synthesis of cellular ATP (Lalla A. et al., West Indian Med. J., 2001 6). Month, 50 (2), pages 111-6. The energy state of the cell is regulated by the AMP-activated protein kinase (AMPK) system and also contributes to the ATP content of the cell (Dagon Y. et al., J. Biol. Chem., 2005, 23, 280 (51), 42142-8).

  The invention also relates, according to a subsequent aspect, to a cosmetic or pharmaceutical or dermatological composition comprising a magnolia champaca oil as described above and a cosmetic or pharmaceutically acceptable medium.

  Preferably, said extract is in a cosmetic, pharmaceutical or dermatological composition in a proportion of 0.001 to 10% of the total weight of the composition, in particular a proportion of 0.01 to 5%, preferably a composition. Present in a proportion of 0.1 to 1% of the total weight of the object.

  Said cosmetic, pharmaceutical or dermatological composition may be particularly suitable for application by the topical route.

  Advantageously, said cosmetic, pharmaceutical or dermatological composition is a powder, emulsion, microemulsion, nanoemulsion, suspension, solution, lotion, cream, aqueous or aqueous / alcoholic gel, foam, serum, aerosol solution Alternatively, it may be provided in the form of a dispersion, or a dispersion of lipid vesicles.

  In the case of an emulsion, the emulsion may be a water-in-oil or oil-in-water emulsion.

  The cosmetic, pharmaceutical or dermatological composition of the present invention also includes a solvent selected according to various ingredients and dosage forms.

  Examples include water (preferably demineralized water), alcohols such as ethanol, or diethylene glycol ethers such as ethoxy diglycol or diethylene glycol monomethyl ether.

  Said cosmetic, pharmaceutical or dermatological composition also comprises at least one additive that is common in the art, such as emollients or wetting agents, gelling and / or thickening agents, surfactants, It may also contain at least one compound selected from oils, active ingredients, dyes, preservatives, antioxidants, chelating agents, organic or inorganic powders, pigments, self-tanning agents, sunscreens, essential oils and perfumes.

In particular, without limitation, the composition may include:
-One or more wetting agents selected from, for example, polyols such as glycerol, propylene glycol, butylene glycol, polyethylene glycol, saccharides or derivatives thereof, urea, glycolic acid or salts thereof, lactic acid or salts thereof, or hyaluronic acid .

The wetting agent will be present in the composition at a content of about 0.01 to 20%, preferably about 0.5 to 10% of the total weight of the composition.
One or more emollients selected from, for example, volatile or non-volatile silicones (dimethicone, cyclomethicone, dimethiconol, and the like), long chain hydrocarbons, or esters of fatty acids or fatty alcohols.

The emollient is present in the composition in a content of about 0.5 to 30%, preferably about 0.5 to 15% of the total weight of the composition.
-Cellulose derivatives, gums derived from plants (guar, locust bean, alginate, carrageenan, pectin), gums derived from microorganisms (xanthan), clays (laponite), carboxyvinyl polymers (carbomers), acrylic acid copolymers, for example Acrylate / alkyl acrylate copolymers, polyacrylamides, ammonium acryloyl dimethyl taurate copolymers such as ammonium acryloyl dimethyl taurate / vinyl pyrrolidone (VP) copolymer and ammonium acryloyl dimethyl taurate / Beheneth-25 methacrylate copolymer (for example, Aristoflex AVC and by Clariant) One or more gels selected from those sold under the name HMB Agents and / or thickeners, preferably one or more hydrophilic gelling agents.

  Non-film-forming gelling agents are, for example, clay (laponite), ammonium acryloyl dimethyl taurate / VP copolymer and ammonium acryloyl dimethyl taurate / beheneth-25 methacrylate copolymer (for example sold by Clariant under the names Aristoflex AVC and HMB) Etc.) may be selected.

The gelling agent and / or thickener is present in the composition in a content of 0 to 10% of the total weight of the composition, preferably about 0.1 to 5% of the total weight of the composition. become.
One or more surfactants or cosurfactants, preferably nonionic, present in the composition in a content of from 0.01 to 10%, preferably from 0.1 to 5% of the total weight of the composition Surfactant.
-Liquid or solid at ambient temperature, usually referred to as oil, may be volatile or non-volatile, may be hydrocarbon-based or silicone-based, and linear , Cyclic or branched or buttery, for example, isododecane, cyclopentadimethylsiloxane, dimethicone, isononyl isononanoate, pentaerythrityl tetraisostearate, caprylic and capric triglycerides, shea butter and the like. One or more fatty substances, preferably in a proportion of 0.01 to about 15%, preferably 0.1 to 10% of the total weight of the composition.
-Natural, with biological activity selected from, for example, vitamins, trace elements, allantoin, plant proteins, plant extracts and the like, and having efficacy on the skin through biological sites, One or more active ingredients derived from biotechnology or synthesis. In particular, the active ingredient may be an algal extract.
-Ponceau disodium salt, alizarin green disodium salt, quinoline yellow, amaranth trisodium salt, tartrazine disodium salt, rhodamine monosodium salt, fuchsin disodium salt, or xanthophyll, preferably One or more water-soluble dyes in a proportion of 0 to about 2% of the total weight of the composition.

  Other additives commonly used in the cosmetic, pharmaceutical or dermatological field may also be present in the compositions of the present invention, and in particular they are preservatives, chelating agents, antiseptics well known in the art. Oxidizing agent, essential oil or fragrance.

  Among all these possible additives, the skilled artisan may select the composition and the amount of additive that will be added to the composition such that the composition maintains all of its properties. You are in a position where you can.

  The invention is illustrated by the following examples without being implicitly limited.

Method for Preparation of Magnolia Champaca Oil of the Present Invention 1) Hexane Extraction 200 kg of fresh magnolia champaca flower is charged into a 1000 L reactor.
Add 800 L of hexane and heat at 35 ° C. for 40 minutes.
Remove the hexane solution from the reactor. The filled solvent is left behind.
600 L of hexane is again charged to the reactor. Heat at 35 ° C. for 40 minutes.
Again, the hexane solution is removed from the reactor. Combine the two filtrates.
Evaporate hexane.
In this way, 0.56 kg of magnolia champaca coagulum is recovered.
Yield: 0.28%

2) Molecular distillation 303 g of Magnolia champaca coagulum is mixed with 130 g of polyethylene glycol 600 (INCI name: PEG 12).

  According to the following distillation parameters, a two-stage distillation is carried out with a KDL4 type (UIC GmH) distillation apparatus:

The yield of distillation is as follows and is expressed relative to the weight of the starting material:
Distillate, 1st stage = 25.7%
Distillate, 2nd stage = 31.34%

3) Mixing After the two distillates are combined and homogenized, a mixture is formed using propylene glycol: 90 wt% propylene glycol is mixed with 10 wt% distillate. The mixture is homogenized and filtered through a 25 μm filter.
Collect the filtrate.
1640 g of magnolia champaca oil is obtained.

Analysis of Magnolia Champaca Oil Composition of the Invention 100 mg of the extract obtained in the third stage of the method described in Example 1 is dissolved in 2 ml of 1N hydrochloric acid methanol solution. The solution is stirred and then placed in an 80 ° C. water bath for 10 minutes.

The sample thus esterified is recovered by liquid-liquid extraction using 2 ml of heptane and injected into gas chromatography.
Chromatography is performed under the following conditions:
Gas chromatograph (Agilent series 6890).
Inject, 0.5 μl of sample prepared by splitting into 1/100 split or splitless injections at 250 ° C.
Separation on 100% grafted polydimethylsiloxane capillary column, length 30 m, inner diameter 0.25 mm, film thickness: 0.25 μm (Agilent series DB-1).
Carrier gas: helium, 1 ml / min Initial temperature of column oven: 70 ° C
Temperature gradient from 70 ° C. to 250 ° C., 2 ° C./min, isothermal at 250 ° C. for 60 minutes.
Total time: 120 minutes Detection: Flame ionization detector (auxiliary gas nitrogen)

  The percentages shown in Table 1 below are given as relative percentages obtained by the chromatographic integrator. This result includes only those compounds that can evaporate in the injector. Propylene glycol is not integrated and is therefore not listed in the table below.

  Components were identified by mass spectrum.

  The results are shown in Table 1 below.

Study on activity of Magnolia champaca oil of the present invention against growth factor HB-EGF Samples of magnolia champaca oil obtained in Example 1 previously diluted to 10% in propylene glycol were tested at various concentrations.

1) Preparation of keratinocyte cultures This protocol is common to all biological activity assays.
Keratinocytes derived from neonatal foreskin (Clonetics, San Diego, USA) were seeded in 6-well plates and keratinocyte growth media (KBM, Clonetics), ie recombinant human EGF, insulin, hydrocortisone, bovine pituitary extract, gentamicin and Cultured in modified medium supplemented with amphotericin B.
After culturing in an incubator at 37 ° C., 5% CO ₂ and saturated humidity for 24 hours, the cells were washed with PBS buffer pH 7.4 (Gibco) and a specific basic medium (KBM containing various concentrations of the test substance). , Clonetics) for 6 or 24 hours depending on the test.
Each concentration was tested in triplicate for two donors for the “ELISA” and “Western Blot” tests and twice for the two donors for molecular biological tests.
Test substance free ("untreated") controls were also tested by maintaining the cells untreated in the same medium.
Positive controls were optionally performed with comparative substances that had been tested for activity.

2) Measurement of HB-EGF messenger RNA (mRNA) expression Assay principle Real-time polymerase chain reaction (RT-PCR) is used to quantify the expression of HB-EGF messenger RNA in treated samples compared to untreated samples. use. Results are normalized for expression of housekeeping genes in these samples and calibrated for differences in PCR efficiency.

  Results are shown as the number of times the expression of the target gene (HB-EGF) is increased or decreased in the treated sample, not as an absolute number of copies.

The cDNA / mRNA sequence of the gene to be investigated was obtained from GenBank.
Target gene: HB-EGF
Housekeeping gene: PBDG

  All PCR primers were obtained using PRIMER3 software from Whitehead Institute for Biomedical Research.

Assay Protocol mRNA was isolated using Trizol reagent (Invitrogen) according to manufacturer's recommendations. Reverse transcription was performed using the GeneAmp RNA PCR kit (Applied Biosystems) according to the manufacturer's recommendations.

Real-time PCR measurements were performed using the LightCycler method (Roche Applied Science) equipped with a SYBR Green I detector. In all assays, cDNA was amplified using a standardized program. Each LightCycler capillary is filled with 1.5 μl DNA master mix, 1.8 μl MgCl ₂ (25 mM), 10.1 μl water and 0.5 μl of each primer (10 μM stock). The final amount of cDNA per reaction corresponds to 25 ng of total RNA used for reverse transcription.

  The specificity of PCR was tested by agarose gel electrophoresis and nucleotide sequence testing, and each sample was evaluated using melting point analysis included in the PCR program.

  Relative quantification of target gene expression was performed using the Pfaffl mathematical model (Pfaffl, MW, Nucleic Acids, Res., 29 (9), E45, 2001).

  The results are shown in Table 2 below.

  As a positive control, tumor necrosis factor α (TNFα), an anti-inflammatory cytokine known to stimulate HB-EGF, was used.

  The results show that the magnolia champaca oil of the present invention has stimulating activity for the synthesis of HB-EGF mRNA.

Study on activity of Magnolia champaca oil of the present invention against growth factor VEGF Measurement was performed by RT-PCR in the same manner as in Example 3. The cDNA / mRNA sequence of the gene to be investigated was obtained from GenBank.
Target gene: VEGF
Culture conditions for housekeeping gene: β ₂ -microglobulin keratinocytes are as described in Example 3.

  The results are reported in Table 3 below:

  The results show that the magnolia champaca oil of the present invention has stimulating activity for the synthesis of VEGF mRNA.

Study of the activity of the magnolia champaca oil of the present invention against the growth factor PDGF-AA Quantitative evaluation of the concentration of human PDGF-AA growth factor in cell culture was performed using a Quantikine® immunoassay kit (No. DAA00, R & D Systems) ) Using the ELISA method.

  Keratinocyte culture conditions and tested samples are as described in Example 3.

  The results are reported in Table 4 below:

  The results show that the magnolia champaca oil of the present invention has stimulating activity on the synthesis of PDGF-AA.

Study on the activity of the magnolia champaca oil of the present invention against endothelin-1 Quantitative evaluation of the concentration of human endothelin-1 in cell culture was performed using a Quantikine® immunology assay kit (No. BBE5, R & D Systems). The ELISA method was used.

  Keratinocyte culture conditions and tested samples are as described in Example 3.

  The results are reported in Table 5 below:

  The results show that the magnolia champaca oil of the present invention has an inhibitory activity on the synthesis of endothelin-1.

Study on the activity of Magnolia champaca oil of the present invention against MITF Quantitative evaluation of the concentration of MITF mRNA in the cell culture was performed according to the protocol described in Example 3.
Target gene: MITF
Housekeeping gene: Kojic acid was used as a positive control for β ₂ -microglobulin inhibition.

  The results are reported in Table 6 below.

  The results show that the magnolia champaca oil of the present invention has an inhibitory activity on the expression of MITF mRNA.

Study of the activity of magnolia champaca oil of the present invention against matrix metalloproteinase (MMP) 1 / Principle of assay The substance to be tested is incubated with activated MMP. Enzyme activity is controlled by adding a fluorescent enzyme substrate specific for each MMP.

2 / Assay Protocol Incubate Pro-MMP3 or Pro-MMP9 with 1 mM APMA (p-aminophenylmercury acetate) solution at ambient temperature for 1 hour with gentle agitation.

  Various potential inhibitors are added at various concentrations. The mixture is then incubated at ambient temperature with gentle agitation. The enzyme reaction is started by adding a fluorescent substrate (SIGMA) dissolved in DMSO.

  The enzyme reaction is monitored for 1 hour using a fluorimeter, with excitation wavelength 360 nm and emission wavelength 460 nm for MMP3 (ie stromelysin 1), excitation wavelength 320 nm and emission wavelength 405 nm for MMP9 (ie gelatinase B). Measure fluorescence.

  The results are expressed in units of emitted fluorescence (RFU), which corresponds to the amount of substrate to be hydrolyzed per minute.

  Absorption variation Δ is calculated with a Microwin 2000 fluorometer. This corresponds to the initial rate (Vi) of the enzyme reaction. In each assay, the average of the three values of Vi obtained for each concentration of potential inhibitor is calculated. The results of 10 experiments were expressed as mean ± SD (standard deviation) and then as a percentage of residual activity.

  The results are reported in Table 7 below (MMP3 or stromelysin 1) and Table 8 (MMP9 or gelatinase B).

  As a positive control, ethylenediaminetetraacetic acid (EDTA) having a well-known inhibitory activity against metalloproteinases was used.

  The results show that the magnolia champaca oil of the present invention inhibits the activity of matrix metalloproteinases MMP3 and MMP9.

Study of the activity of the Magnolia champaca oil of the present invention on the synthesis of DT diaphorase After preparing the cells according to the protocol of Example 3, the cells are lysed for analysis by Western blotting. After quantifying the protein according to the kit (Pierce Chemical Co., New York), the protein is fractionated on a gel gradient. Polyvinylidene difluoride (PVDF) membranes resulting from transfer from the gel are incubated in the presence of anti-NQO1 polyclonal antibody (Genetex, San Atonia, TX). After rinsing, the membrane is developed with the “Supersignal West Dura” detection kit (Pierce Chemical Co., New York).

  The results are reported in Table 9 below.

  The results show that the magnolia champaca oil of the present invention stimulates the activity of DH diaphorase.

Study on the activity of the Magnolia champaca oil of the present invention on the synthesis of glutathione S-transferase (GST) After preparing cells according to the protocol of Example 3, the cells are lysed and the supernatant is recovered. The activity of GST is assessed according to the protocol described by the kit supplier by using a “GST activity assay kit” (Biodivision, Mountain View, Calif.).

  The results are reported in Table 10 below:

The results show that the magnolia champaca oil of the present invention stimulates the synthesis of glutathione S-transferase.

Study of the activity of the magnolia champaca oil of the present invention on the synthesis of cellular cytochrome C The amount of cytochrome C in the lysate of keratinocytes prepared according to the protocol of Example 3 was determined using the “cytochrome C ELISA kit” (Zymed®). Quantify using.

  Results are reported in Table 11 below:

  The results show that the magnolia champaca oil of the present invention stimulates the synthesis of cellular cytochrome C.

Study of the activity of the Magnolia champaca oil of the present invention on the synthesis of AMP kinase After preparing the cells according to the protocol of Example 3, the cells are lysed for analysis by Western blotting. After quantifying the protein according to the kit (Pierce Chemical Co., New York), the protein is fractionated on a gel gradient. Membranes resulting from transfer from gel to membrane (PVDF) are incubated in the presence of anti-AMPK or anti-phospho-AMPKα monoclonal antibody (Cell Signaling Technology, Beverly, Mass.). After rinsing, the membrane is developed with the “Supersignal West Dura” detection kit (Pierce Chemical Co., New York) and the bands are quantified by image analysis.

  The results are reported in Table 12 below:

  The results show that the magnolia champaca oil of the present invention has stimulating activity for the synthesis of AMP kinase.

Study of Antioxidant Activity of Magnolia Champaca Oil of the Invention Three Protocol Principles The ability to measure the ability of a sample to block free radicals can be determined by various Abel® 1 Antioxidant Test Kits (Knight Scientific Limited) Is possible. In each experiment, a specific type of radical or oxidant is generated: a hydroxyl radical, a halogenated oxidant or a superoxide radical. These chemorminescent tests include a special Pholasin® photoprotein that emits light in the presence of free radicals and certain oxidizing agents. The antioxidant capacity of a sample is represented by the percentage decrease in the emission peak of Pholasin® observed during various tests in the presence or absence of the sample or control.

  A positive control was used for each of the following tests a, b and c.

a / Activity studies on hydroxyl radical inhibition of magnolia champaca oil of the present invention As a positive control, mannitol which exerts a known anti-free radical effect on hydroxyl radical was used.

  The results show that the magnolia champaca oil of the present invention has antioxidant activity against hydroxyl radicals.

b / Study on activity of halogenated oxidant inhibition of magnolia champaca oil of the present invention As a positive control, bovine serum albumin (BSA) exhibiting known inhibitory activity against halogenated oxidant was used.

The results show that the magnolia champaca oil of the present invention has antioxidant activity against halogenated oxidants.

c / Study of superoxide radical inhibition activity of magnolia champaca oil of the present invention Vitamin C (ascorbic acid) was used as a positive control. Ascorbic acid is well known for its role in the formation of superoxide radicals.

  The results show that the magnolia champaca oil of the present invention has an antioxidant activity against superoxide radicals.

ICON TOA CREAM CONTAINING MAGNORIA CHAMPAKA OIL OF THE INVENTION The various ingredients listed in Tables 16 and 17 below may be referred to by their general chemical names or by the INCI International Nomenclature Fragrance Association Publication, 9th edition, 2002).

  Face cream containing magnolia champaca oil of the present invention

Claims (36)

20 to 70% benzoic acid esters,
15 to 35% saturated and / or unsaturated fatty acids (the ratio is expressed as a relative percentage of the sum of the components separated by gas chromatography)
Oil extracted from at least one Magnolia champaca flower characterized in that   2. The oil according to claim 1, wherein the oil is derived from magnolia champaca and variety gold. -10 to 40% cinnamyl benzoate,
-5-30% phenylethyl benzoate,
5 to 30% benzyl benzoate,
-5 to 30% linoleic acid,
-2 to 20% linolenic acid (the ratio is expressed as a relative percentage of the sum of the components separated by gas chromatography)
Oil according to claim 1 or 2, characterized in that   Oil according to any one of claims 1 to 3, characterized in that it does not contain a solvent.   Oil according to any one of claims 1 to 4, characterized in that it does not contain any chemical reactants involved in the extraction process.   6. A process for preparing magnolia champaca oil according to any one of claims 1 to 5, characterized in that it comprises at least one molecular distillation stage. -Extraction with at least one non-polar solvent from the ground product produced from fresh flowers of at least one magnolia champaca;
Magnolia champaca oil according to any one of claims 1 to 5, characterized in that it comprises:-subjecting said extract to at least one molecular distillation step; and-recovering the distillate. Method for preparing the. Wherein the non-polar solvent, wherein hexane, cyclohexane, heptane, isooctane, to be selected from supercritical CO ₂ or dichloromethane, The method of claim 7.   9. The molecular distillation step according to any one of claims 6 to 8, characterized in that the molecular distillation step is carried out at a temperature between about 100 <0> C and 200 <0> C and a pressure between about 10 and 0.001 millibar. The method according to one item. During distillation completion, fractionation, characterized in that it is carried out with C ₁ -C ₄ alcohols, polyols, ethyl acetate, hexane, at least one solvent selected from cyclohexane and supercritical CO _2, claim The method according to any one of 7 to 9.   11. Process according to any one of claims 6 to 10, characterized in that a solvent selected from mineral or vegetable oils and polyols is added prior to molecular distillation.   12. The temperature according to any one of claims 6 to 11, characterized in that the temperature at the introduction to the distillation column is between 20 and 120 ° C, preferably between 50 and 100 ° C. Method.   13. Process according to any one of claims 6 to 12, characterized in that the concentration temperature is between 40 <0> C and 120 <0> C, preferably between 60 <0> C and 100 <0> C.   Magnolia champaca oil obtainable by the method according to any one of claims 6 to 13.   As a multifunctional active ingredient in pharmaceutical, cosmetic or dermatological compositions for preventing and / or treating aging or physiological mechanisms associated with aging or diseases associated with these mechanisms Use of magnolia champaca oil according to any one of claims 1 to 5 or claim 14.   Agents that improve healing or cell repair and / or agents that activate epidermal cell metabolism and / or agents that trap free radicals and / or agents that regenerate the epidermis and / or detoxify the epidermis Use according to claim 15 of magnolia champaca oil according to any one of claims 1 to 5 or claim 14 in a cosmetic composition as a medicament for.   Agents for improving healing or cell repair and / or agents for activating epidermal cell metabolism and / or agents for scavenging free radicals and / or agents for protecting nuclear DNA and / or mitochondrial DNA 15. Any of claims 1 to 5 or claim 14 for the preparation of a pharmaceutical composition as an agent for protecting and / or an agent for regenerating the epidermis and / or an agent for detoxifying the epidermis. Use according to claim 15 of magnolia champaca oil according to one.   Agents for improving healing or cell repair and / or agents for activating epidermal cell metabolism and / or agents for scavenging free radicals and / or agents for protecting nuclear DNA and / or mitochondrial DNA 15. Any of claims 1 to 5 or claim 14 for the preparation of a dermatological composition as an agent for protecting and / or an agent for regenerating the epidermis and / or an agent for detoxifying the epidermis Use of magnolia champaca oil according to claim 15.   19. Use according to any one of claims 15 to 18 of magnolia champaca oil as a drug that inhibits melanin synthesis.   20. Use of magnolia champaca oil according to any one of claims 15 to 19 as an agent that inhibits endothelin synthesis or an agent that inhibits the transcription factor MITF.   20. The use according to any one of claims 15 to 19 of magnolia champaca oil as an agent that activates the synthesis of at least one cell growth factor by keratinocytes.   Use according to claim 21, characterized in that the growth factor is selected from PDGF, VEGF and HB-EGF.   19. Use according to any one of claims 15 to 18 of magnolia champaca oil as an agent that inhibits the activity of matrix metalloproteinases MMP3 (ie stromelysin 1) and MMP9 (ie gelatinase-elastase B).   19. Use according to any one of claims 15 to 18 of magnolia champaca oil as a drug that stimulates cellular metabolism.   Use according to claim 24, characterized in that the factor for stimulating cellular metabolism is selected from cytochrome C or AMP kinase.   19. Use according to any one of claims 15 to 18 of magnolia champaca oil as an antioxidant.   Use according to any one of claims 15 to 18 of magnolia champaca oil as a medicament for detoxification of the epidermis by stimulating the synthesis of DT diaphorase.   A cosmetic, pharmaceutical or dermatological composition comprising a magnolia champaca oil according to any one of claims 1 to 5 or claim 14 and a cosmetic or pharmaceutical acceptable medium.   30. Cosmetic, according to claim 28, characterized in that the oil is present in a pharmaceutical, cosmetic or dermatological composition in a proportion of 0.001 to 10% of the total weight of the composition. Pharmaceutical or dermatological composition.   Said oil is present in the pharmaceutical, cosmetic or dermatological composition in a proportion of 0.01 to 5%, preferably 0.1 to 1% of the total weight of the composition, 30. A cosmetic, pharmaceutical or dermatological composition according to claim 29.   31. Cosmetic, pharmaceutical or dermatological composition according to any one of claims 28 to 30, characterized in that it is suitable for application by the topical route.   Emollient or wetting agent, gelling agent and / or thickener, surfactant, oil, active ingredient, dye, preservative, antioxidant, chelating agent, organic or inorganic powder, pigment, self-tanning agent, sunscreen The cosmetic, pharmaceutical or dermatological composition according to any one of claims 28 to 31, characterized in that it also comprises at least one compound selected from essential oils and perfumes.   The cosmetic or pharmaceutical product according to claim 32, characterized in that it comprises a natural, biotechnological or synthetically active active ingredient that has biological activity and has an effect on the skin through a biological site. Or dermatological composition.   34. Cosmetic, medicinal or skin according to any one of claims 32 and 33, characterized in that the active ingredient is selected from vitamins, trace elements, allantoin, plant proteins, plant extracts and mixtures thereof. Scientific composition.   35. A cosmetic, pharmaceutical or dermatological composition according to one of claims 32 to 34, characterized in that the active ingredient is an algal extract.   Provided in the form of a powder, emulsion, microemulsion, nanoemulsion, suspension, solution, lotion, cream, aqueous or aqueous / alcoholic gel, foam, serum, aerosol solution or dispersion, or lipid vesicle dispersion 36. A cosmetic, pharmaceutical or dermatological composition according to any one of claims 28 to 35, characterized in that